Rapid Enhancement of Chick Intestinal DNA-Dependent RNA Polymerase II Activity by 1α,25-Dihydroxyvitamin D ₃ , In Vivo

Abstract

la,25-dihydroxyvitamin D3 was examined for its ability to affect the DNA-dependent RNA polymerases (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of rachitic chick intestinal cell nuclei in vivo. Nucleoplasmic (form II) RNA polymerase activity was stimulated 2-fold (P < 0.05) within 2-3 hr after an oral dose of 0.27 sAg (0.65 nmol) of la,25-dihydroxyvitamin D3 to rachitic chicks. The form II polymerase activity returned to control values by 5-9 hr after dosing with the sterol. In contrast, the nucleolar (form I) RNA polymerase was not increased within this period. Solubilization of nuclear protein and resolution of the two RNA polymerases on DEAE-Sephadex also revealed that there was an increase in polymerase II activity when assayed on exogenous DNA template. This evidence suggests that la,25-dihydroxyvitamin D3 acts at the level of the enzymology of intestinal cell transcription and that increased mRNA synthesis after administration of this hormone cannot be due solely to a change in chromatin template activity.It is now well established that vitamin D3 is converted to 25-hydroxyvitamin D3 (25-OH-D3) in several tissues (1, 2) and that this metabolite is again hydroxylated to la,25-dihydroxyvitamin D3 [la, 25-(OH)2-D] in the kidney (3). la,25-(OH)2-D3 has been shown to be the most active metabolite of vitamin D3 in stimulating intestinal calcium absorption (4) and bone calcium resorption (5, 6), and it is considered to be the physiologically active form of vitamin D3. Studies using radioactive vitamin D3 have shown that the active metabolite functions by localizing in the nucleus of target cells (7,8). More recently, it has been shown that la,25-(OH)2-D3 binds initially to a specific cytosol receptor in the intestine (9) and is then transported to the nucleus where it becomes associated with the chromatin (10-13). Furthermore, the la,25-(OH)rDr mediated stimulation of intestinal calcium absorption is inhibited by actinomycin D in vivo (14) and in vitro (15). Recent evidence suggests that vitamin D3 and its active metabolite may modulate the synthesis of a specific mRNA which codes for the de novo synthesis of calcium-binding protein (16), a functional mediator of calcium translocation in the intestine (17). This evidence indicates that la,25-(OH)rD3 may be functioning at the level of gene transcription, similar to the steroid hormones (18,19).Further support for the view that la,25-(OH)rD3 stimulates calcium absorption by regulating specific mRNA synthesis has been demonstrated in vitro using a-amanitin (15), the specific inhibitor of nucleoplasmic DNA-dependent RNA polymerase II (20,21). Since a-amanitin inhibits the induction of a number of enzyme activities in eukaryotic systems (22-24), we decided to determine the effect of la,25-(OH)2-D3 in vivo on the transcriptive enzymes of mucosal nuclei as assayed in vitro. Evidence is presented in this report that la, 25-(OH)2-D3 significantly stimulates RNA polymerase II activity in the target tissue after saturation of mucosal cell nuclei ...

Stimulation of intestinal calcium-binding-protein mRNA synthesis in the nucleus of vitamin D-deficient chicks by 1,25-dihydroxycholecalciferol

“…3). Yet the amount of hormone accumulated in the nucleus (Lawson & Emtage, 1974), together with the increased RNA polymerase II activity (Zerwekh et al, 1974), are maximal within 2h. In other systems, newly transcribed mRNA is processed very rapidly, with a halflife of minutes, into mature mRNA (Curtis & Weissmann, 1976;Bastos & Aviv, 1977).…”

“…It was found that after the rapid accumulation of the hormone in chick intestinal nuclei (Lawson & Emtage, 1974) nuclear RNA synthesis is stimulated (Tsai & Norman, 1973), as is RNA polymerase activity (Zerwekh et al, 1974) and DNA template capacity (Zerwekh et al, 1976), and the physiological response is inhibited in vitro by a-amanitin and actinomycin D (Corradino, 1973). Thus RNA transcription and protein synthesis were implicated in the mechanism ofaction ofthe hormone.…”

“…Since this is in agreement with the time by which the most hormone has accumulated in the nucleus (Lawson & Emtage, 1974) Vol. 175 and with the reported increase in RNA polymerase II activity in the nucleus (Zerwekh et al, 1974), it has been assumed by many workers that the transcription and translational events referred to above were those required for the synthesis of calcium-binding protein.…”

Enhancement of soybean RNA polymerase I by auxin.

“…DISCUSSION A wide variety of hormones administered in vivo have been reported to result in enhanced chromatin-bound or nuclear RNA polymerase in both plants (3,13,14) and animals (15)(16)(17)(18). Both nucleolar (17,18) and nucleoplasmic (19) RNA polymerases are subject to hormonal regulation in animals. The nucleolar RNA polymerase (RNA polymerase I) transcribes ribosomal RNA (20,6), while the nucleoplasmic RNA polymerase (RNA polymerase II) is thought to transcribe messenger RNA (21).…”

Decreased intestinal calcium absorption in vivo and normal brush border membrane vesicle calcium uptake in cortisol-treated chickens: evidence for dissociation of calcium absorption from brush border vesicle uptake.

“…It is known, for example, that 1,25-(OH)2D3 is rapidly localized to the nucleus of intestinal cells (4)(5)(6)(7)(8). In all likelihood, 1,25-(OH)2D3 enters the cell and associates with a cytosolic binding protein (9,10) prior to translocation into the nucleus (11). In the nucleus it increases RNA polymerase II activity (11), RNA synthesis (12), chromatin template activity (13), and the synthesis of mRNA for calcium-binding protein (14).…”